KR101075030B1

KR101075030B1 - biochip

Info

Publication number: KR101075030B1
Application number: KR1020090050108A
Authority: KR
Inventors: 김동현; 심은지; 김규정; 최종률
Original assignee: 연세대학교 산학협력단
Priority date: 2009-06-05
Filing date: 2009-06-05
Publication date: 2011-10-21
Also published as: KR20100131291A

Abstract

Biochip according to an aspect of the present invention, the glass substrate; A metal lattice / thin film layer formed on a glass substrate to support a sample including fluorescent molecules and having a nanolattice structure on the top thereof; A plate member for cell culture disposed on the metal lattice / thin film layer and in which at least one cell culture chamber is formed; And a microchannel plate member disposed on the cell culture plate member and having a through opening communicating with the cell culture chamber, and having a microchannel connected to the through opening for supplying a fluid to the cell culture chamber. The metal lattice / thin film layer may include a metal thin film; And a metal lattice formed on the metal thin film to have a nano lattice structure having a constant lattice thickness (d _g ) and a constant lattice period (A) smaller than the wavelength of the incident light to generate local surface plasmon resonance for the incident light. have.

Total reflection fluorescence microscope

Description

Biochip {biochip}

The present invention relates to a total reflection fluorescence microscope used as an optical measuring device for a biological sample (sample) such as a cell and a biochip applicable thereto. Total reflection fluorescence microscope that can locally optimize or maximize the vanishing wave generated in the total reflection shape by using the principle of surface plasmon resonance to improve the sensitivity of the total reflection fluorescence microscope, and the optical arrangement structure for maximizing the vanishing wave It relates to a biochip that can be applied to a total reflection fluorescence microscope.

A total internal reflection fluorescence microscope (TIRF microscope), which is an optical measurement method, is used to obtain a fluorescence information about the local depth of a biological sample by a total wave incident from the total reflection by injecting it at a total reflection angle on the biological sample marked with a fluorescent label. have. Such total reflection fluorescence microscopy is mainly used for fluorescence observation of biological molecules in cell biology, medicine, pharmaceutical or biochemistry, and is often used to observe fluorescence images from biological samples such as single molecules or cells.

A conventional total reflection fluorescence microscope has a basic configuration in which an fluorescence is generated from fluorescent molecules in a sample layer by total reflection of incident light at an interface between the sample layer and glass (substrate) in an optical arrangement structure that generates fluorescence from a sample. have. However, high detection sensitivity is required to observe fluorescence of localized regions of molecular level particles or biological samples. Therefore, as a light source used for the total reflection fluorescence microscope, a light source with a relatively high intensity is required, and the fluorescence detection unit requires a high sensitivity CCD camera or the like. Even when such a highly sensitive and expensive equipment is used, it may be difficult to accurately observe fluorescence in a local region of a biological sample such as a cell. Accordingly, it is necessary to further improve the detection sensitivity of a total reflection fluorescence microscope.

One object of the present invention is to provide a total reflection fluorescence microscope which can improve the sensitivity of fluorescence detection for a sample such as cells and thereby obtain more meaningful biological information.

Another object of the present invention is to provide a biochip that can be effectively applied to a total reflection fluorescence microscope to improve the optical measurement sensitivity by the total reflection fluorescence microscope.

A total reflection fluorescence microscope according to an aspect of the present invention, a prism; A metal lattice / thin film layer formed on the prism to support a sample including fluorescent molecules and having a nanolattice structure on the top; A light source unit providing incident light at which total reflection occurs at an interface between the metal lattice / thin film layer and a sample; And a fluorescence detector for detecting fluorescence generated by vanishing waves emitted by the total reflection near the interface. The metal grating / film layer has a metal film and a metal grating. The metal grating is formed on the metal thin film, and has a nano lattice structure having a constant grating thickness (d _g ) and a constant grating period (A) smaller than the wavelength of the incident light to generate local surface plasmon resonance for the incident light. .

According to the exemplary embodiment of the present invention, the metal lattice may have a plurality of stripe-shaped lattice structures arranged parallel to each other on the metal thin film. The metal thin film and the metal lattice may be formed of the same metal material. The metal lattice / thin film may be formed of silver or gold. The light source unit may use a helium-cadmium laser light source. The total reflection fluorescence microscope may further include a glass substrate disposed between the prism and the metal lattice / thin layer.

According to an embodiment of the present invention, the lattice thickness d _g of the metal lattice is 10 to 20 nm, the thickness d _f of the metal thin film is 10 to 20 nm, and the lattice period A of the metal lattice May be from 100 to 300 nm.

Biochip according to another aspect of the invention, the glass substrate; And a metal lattice / thin layer formed on the glass substrate to support a sample including fluorescent molecules and having a nanolattice structure on the top thereof. The metal lattice / thin film layer has a metal thin film and a metal lattice. The metal grating is formed on the metal thin film, and has a nano lattice structure having a constant grating thickness (d _g ) and a constant grating period (A) smaller than the wavelength of the incident light to generate local surface plasmon resonance for the incident light. .

According to an embodiment of the present invention, the biochip comprises: a cell culture plate member disposed on the metal lattice / thin film layer and having at least one cell culture chamber; And a micro-fluidic channel disposed on the cell culture plate member, the through opening communicating with the cell culture chamber, and connected to the through opening for supplying a fluid to the cell culture chamber. It may further comprise a plate for the fine flow path formed.

In addition, the biochip, the lower glass cover plate disposed under the glass substrate; And an upper glass cover plate disposed on the fine flow path plate member. The upper glass cover plate is formed with an inlet hole for communicating with the microchannel and for supplying a fluid to the cell culture chamber, and an outlet hole for discharging the fluid that has communicated with the microchannel and passed through the cell culture chamber. It may be formed.

In addition, the biochip may further include a close contact plate member disposed between the upper glass cover plate and the fine flow path plate member. The close contact plate member serves to closely contact the upper glass cover plate and the fine flow path plate member. The close contact plate member has a hole communicating with the inlet hole and the microchannel, and a hole communicating with the outlet hole and the microchannel.

According to the present invention, it is possible not only to obtain high fluorescence detection sensitivity as compared to a conventional total reflection fluorescence microscope system, but also to obtain fluorescence information with higher sensitivity without changing detection units such as a light source and a CCD camera of a conventional total reflection fluorescence microscope. have. Accordingly, the total reflection microscope and the biochip according to the embodiments of the present invention can be effectively used for biological, biochemical, and medical research. In addition, in-situ experiments on biological samples such as living cells can be performed using a nanochip integrated biochip with high measurement sensitivity, thereby providing a lap-on-a-chip. Efficient application is possible.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. The shape and the size of the elements in the drawings may be exaggerated for clarity and the same elements are denoted by the same reference numerals in the drawings.

According to the embodiment of the present invention, in order to improve the fluorescence detection sensitivity of the total reflection fluorescence microscope, in the optical arrangement structure using the principle of surface plasmon resonance, the vanishing wave generated in the total reflection phenomenon can be locally maximized and optimized. A metal nanogrid is placed between the sample (sample) and the prism. Depending on the type of metal, optical modeling and optimized grating periods and grating thicknesses can be calculated through rigorous coupled wave analysis (RCWA) or finite difference time domain (FDTD) simulations. It was confirmed. Patterning methods used in semiconductor processes such as electron beam lithography can be used to form nanoscale grating periods and grating thicknesses.

1 is a diagram schematically showing a total reflection fluorescence microscope according to an embodiment of the present invention. Referring to FIG. 1, the total reflection fluorescence microscope has an optical arrangement structure 50 in which light (for example, laser light) from the light source unit 60 is incident to total reflection. A sample 55 such as a biological sample such as a cell is loaded on the optical arrangement structure 50. For example, helium-cadmium laser light having a wavelength of 442 nm is emitted from the light source unit 60 and passes through a collimating lens and a beam expander 62, and the p-mode (TM) by the polarizer 64. Mode) is polarized. Reflective mirror 66 may be used to obtain a constant angle of incidence for total reflection. The TM polarized laser light incident on the prism 20 of the optical arrangement structure 50 passes through the glass substrate 40 in the metal thin film 31 having the metal nanograting 33 structure formed thereon. Fluorescence is generated from the fluorescent particles (fluorescent particles in the sample) that are totally reflected (or at the interface between the sample solution and the glass substrate 40) and excited by the near field (disappearance wave) near the interface. The generated fluorescence may be detected in the form of a two-dimensional image by the fluorescence detector 80 having, for example, a CCD camera or the like through an objective lens 70 and a band pass filter (not shown).

Surface plasmon resonance may occur in the metal lattice / thin layers 33 and 31 through incident light having a specific incident angle incident through the prism 20 into the metal thin film 31. In particular, due to the structure of the metal nanolattice 33 having a size smaller than the wavelength of the light source (lattice period and lattice thickness), the evanescent wave or evanescent field resulting from the total reflection is locally maximized and lost on average. The field augmentation effect can be obtained. As a result, the efficiency of generating fluorescence can be maximized and the detection sensitivity of biological samples such as cells or other samples can be greatly improved.

In the total reflection fluorescence microscope 10 of FIG. 1, the SF10 substrate can be used as the prism 20 and the glass substrate 40. Silver or gold may be used as the metal lattice / thin layers 33 and 31, and preferably, the silver lattice / thin layers may be used. The thin metal film and the metal lattice may be made of the same metal material in terms of manufacturing process and loss field reinforcement.

By further disposing an index matching liquid or gel between the prism 20 and the glass substrate 40, the refractive index mismatch between the prism 20 and the glass substrate 40 is matched or the prism 20 ) And the refractive index mismatch due to an air gap that may exist between the glass substrate 40 and the glass substrate 40. In the above-described optical disposition structure 50, the glass substrate 40 is omitted, and the prism ( 20) metal lattice / thin layers 33, 31 may be deposited directly.

2 is a cross-sectional view illustrating an optical arrangement structure using a metal nanogrid to improve sensitivity of fluorescence measurement according to an exemplary embodiment of the present invention. This optical arrangement structure 50 can be placed under the objective lens 70 and irradiated with illumination light, such as laser light, as described above with reference to FIG. Surface plasmon resonance may be generated by total reflection of light incident at the incident angle θ. In particular, the fluorescence emission is enhanced by the propagating plasmon, and the fluorescence emission is further enhanced by the metal lattice structure.

The optical arrangement structure 50 includes a prism 20 and a metal grating / thin layer 30 formed thereon. The metal lattice / thin film layer 30 includes a metal thin film 31 having a predetermined thickness d _f and a metal lattice 33. The metal grating 33 has a nanogrid structure having a constant grating thickness d _g and a constant grating period A. The grating thickness d _g and the grating period A are smaller than the light wavelength of the light source portion. The metal lattice 33 may be formed of the same metal material as the metal thin film 31, preferably formed of silver or gold, and more preferably made of silver. In this embodiment, the metal lattice 33 has a plurality of stripe-like (planar) lattice structures arranged parallel to each other (see Fig. 6), and is cycled on the metal thin film 31 as shown in the cross-sectional view of Fig. 2. To form an overhang.

By using the metal lattice / thin film layer 30 having such a nanolattice structure, it is possible to generate localized surface plasmon resonance for incident light, especially in the vicinity of the edge of the metal lattice, where the plasmon is locally strongly localized. The intensity of the missing wave (or the strength of the missing field) is locally maximized at. This enhances the plasmon excitation efficiency and maximizes the disappearance field at locations near the edge of the periodically arranged grating to increase the fluorescence emission efficiency of the sample.

A dielectric layer, such as a glass substrate (see 40 at FIG. 1) may be further disposed below the metal thin film 31, but it is found that such a dielectric layer does not have a significant effect on the overall properties. Thus, as shown in FIG. 2, the additional glass substrate in the optical arrangement 50 may be omitted.

For the metal lattice / thin layer 30 of the material to be specifically applied, the optimum lattice period (A) and lattice thickness (d _g ) for local surface plasmon resonance (or to maximize the detection sensitivity of the total reflection fluorescence microscope) And the thickness d _f of the metal thin film may be calculated through a rigid coupled wave analysis (RCWA) or finite difference time domain (FDTD) simulation analysis. In particular, the optimum metal thin film thickness (d _f ) can be determined from the near field profile of the nanogrid structure calculated by the FDTD method. The optimal grating thickness dg and grating period A can also be calculated by RCWA or FDTD. According to the embodiment, the lattice thickness d _g of the metal lattice 31 is 10 to 20 nm, the lattice period A is 100 to 300 nm, and the thickness d _f of the metal thin film 33 is 10 to 10 nm. 20 nm.

3 is a graph showing reflectance according to an incident angle of light (laser light) incident on an optical arrangement structure in embodiments of the present invention. The graph of FIG. 3 shows the incident angle where the surface plasmon excitation due to total reflection is greatest when the gold or silver lattice 33 has a nano lattice structure of different lattice periods A and lattice thickness d _g by RCWA simulation. Show that you can calculate In each of the reflectance curves shown in Fig. 3, the largest intensity vanishing wave occurs at the minimum and the strongest surface plasmon excitation occurs. In the embodiments of FIG. 3, the thickness d _f of the metal thin film (which is made of the same metal material as the metal lattice) is all 10 nm, and the fill factor of the metal lattice is fixed at 67%. Referring to FIG. 3, silver gratings having a grating thickness (d _g ) of 20 nm and grating periods (A) of 300 nm have smaller reflectances (ie, more resonance at resonance) than silver gratings having d _g = 10 and A = 300 nm. High plasmon excitation efficiency) and a larger maximum disappearance field. By this enhanced localization, the missing field becomes more spatially non-uniform and localized.

FIG. 4 shows the magnetic field intensity of the near-field calculated by FDTD for various metal grating / thin layers 30, with metals having different materials, grating periods and grating thickness conditions. The lattice / thin layer 30 shows the magnetic field strength of the vanishing wave resulting from the total reflection phenomenon.

4 (a) shows only a prism without the metal lattice / thin layer 30, as in the conventional TIRF, FIG. 4 (b) shows a lattice period A of 300 nm and a metal thin film thickness of 10 nm d _f ), in the case where the gold nano-grid having a grid thickness (d _g) of 10 nm, FIG. 4 (c) is a metal thin film thickness of the grating period (a) of 300 nm and 10 nm (d _f), a grid of 20 nm If the gold nano-grid has a thickness (d _g), Fig. 4 (d) is the grating period (a) and the metal thin film thickness of 10 nm (d _f), of 10 nm grating thickness (d _g) of 100 nm 4 (e) shows a silver nanogrid having a lattice period (A) of 300 nm, a metal thin film thickness (d _f ) of 10 nm, and a lattice thickness (d _g ) of 10 nm. 4 (f) is a result of the disappearance wave when there is a silver nano lattice having a lattice period A of 300 nm, a metal thin film thickness d _f of 10 nm, and a lattice thickness d _g of 20 nm. Indicates the strength of the magnetic field. In FIG. 4, the metal thin film is the same material as the metal lattice.

As shown in Fig. 4, the silver lattice with d _g = 20 nm and A = 300 nm (Fig. 4 (f)) is higher than the silver lattice with d _g = 10 nm and A = 300 nm (Fig. 4 (e)). It shows a larger maximum loss field and is more strongly localized (unevenly). This is exactly what is expected in the reflectance graph of FIG. 3. However, the silver lattice (FIG. 4 (e)) with d _g = 10 nm and A = 300 nm is not only at the lattice edge vicinity P1, which is the maximum field position, but also at the lattice ridge point P2 (ie, the center of the lattice protrusion). It shows a significant strengthening of the lost field. In fact, biological samples such as cells make good contact with the lattice ridge portion substantially, and therefore, it is desirable to consider not only the maximum field position but also the loss field reinforcement at the lattice ridge portion, in determining the optimum dg and A values.

5 is a graph showing a profile of the magnetic field strength (│Hy│) calculated by the FDTD. Referring to FIG. 5, the vanishing wave generation surface position (with and without a silver thin film thickness (10 nm) with the same lattice period (300 nm) and with a silver nano lattice having different lattice thicknesses (10 nm and 20 nm)) The profile of the magnetic field (Hy) intensity according to the y-axis distance from P, P1, P2 is shown.

In Fig. 5, curve a is a profile by the conventional TIRF microscope shown in Fig. 4A (when there is only a prism without a metal lattice / thin layer), in the y-axis direction at one position P on the surface of the prism. Indicates the magnetic field strength over distance. Curve e1 represents the magnetic field strength according to the distance in the y-axis direction from the maximum field point P1 of the silver lattice of d _g = 10 nm and A = 300 nm shown in Fig. 4 (e), and curve e2 is the same. Denotes the magnetic field strength according to the y-axis distance at the ridge point P2 of the lattice. Further, curves f1 and f2 are in the y-axis direction at the maximum field point P1 and the ridge point P2 of the silver grating (d _g = 20 nm, A = 300 nm) shown in FIG. 4 (f). Indicate the magnetic field strength according to the distance.

As shown in FIG. 5, when there is a silver nanolattice, the magnetic field strength of the missing field is greater at the lattice edge P1 than the lattice ridge point P2 (ie, the missing field is localized near the edge). Also, the larger the grating thickness d _g , the greater the field reinforcement or augmentation effect appears at the maximum field point P1. That is, in the penetration depth range, the curve f1 (when dg = 20 nm) exhibits a higher magnetic field intensity than the curve e1 (when dg = 10 nm). However, a larger lattice thickness (dg = 20 nm) does not mean an optimization condition, which means that stronger localization may weaken the field in areas where the field is not localized (i.e. the lattice ridge region). Because. Thus, as shown in FIG. 5, the curve f2 may be located below the curve a. As a result, in view of the overall consideration including the maximum field point P1 and ridge point P2 of the nano lattice, the optimal lattice structure may be determined as a silver lattice structure with dg = 10 nm and A = 300 nm.

FIG. 6 is a photograph (SEM photograph) of an image of a silver nano lattice having a lattice thickness of 10 nm, a silver thin film thickness of 10 nm, and a lattice period of 300 nm formed through electron beam lithography using a scanning electron microscope. In order to form such a nano lattice structure, for example, by depositing silver (more broadly metal) on a glass substrate or prism to form a silver thin film 31, the photoresist PR on the silver thin film 31 A layer is formed and the PR layer is patterned by electron beam lithography to define the grating pattern. After depositing silver on the silver thin film 31 on which the PR lattice pattern is formed, the silver lattice 33 may be formed on the silver thin film 31 by lift off.

FIG. 7 is a photograph obtained by measuring an atomic force microscope (AFM) of an image of a silver nanogrid having a lattice thickness of 10 nm, a silver thin film thickness of 10 nm, and a lattice period of 300 nm formed through electron beam lithography. In Fig. 7, polymer residues scattered on the lattice surface are shown. 6 and 7, the metal lattice 33 of the metal lattice / thin film layer 30 may have a lattice structure of a plurality of stripe shapes (lattice wires) arranged in parallel with each other. 8 is a profile extracted from the image data of FIG. 7 measured through an atomic force microscope.

FIG. 9 is an image of fluorescence microbeads (diameter φ = 1.00 μm ) measured by a total reflection fluorescence microscope using a prism without applying a metal nanogrid according to a conventional example. 10 is a silver nano lattice structure having a lattice thickness of 10 nm, a silver thin film thickness of 10 nm, and a lattice period of 300 nm under the same conditions (except for the nano lattice structure) as in the experiment of FIG. 9 according to an embodiment. When measured, it is an image of fluorescence microbeads (diameter ( φ ) = 1.00 μm ) measured with a total reflection fluorescence microscope. 9 and 10, it can be seen that the detection sensitivity of the fluorescent microbeads is much improved when the optical arrangement structure using the metal nanogrid according to the embodiment is used.

FIG. 11 is an electron scanning microscope (SEM) photograph of cells cultured on a silver nanogrid structure having a lattice thickness of 10 nm, a silver thin film thickness of 10 nm, and a lattice period of 300 nm. 12 is an SEM photograph obtained by enlarging the electron scanning microscope magnification of FIG. 11. The cells shown in FIGS. 11 and 12 are A431 human epithelial cancer cells, which are in good contact with the lattice surface as shown. Therefore, even if the sample is a biological sample such as a cell, it can be seen that such a sample (cell, etc.) can be cultured on the above-described metal nano lattice structure and adhere well, and can be usefully applied to analytical experiments of various biological samples. .

FIG. 13 is an image obtained by measuring a quantum dot applied to A431 cells by a total reflection fluorescence microscope using a prism without applying a metal nanogrid according to a conventional example. FIG. 14 is a total reflection fluorescence of a quantum dot applied to A431 cells when a silver nanolattice structure having a lattice thickness of 10 nm, a silver thin film thickness of 10 nm, and a lattice period of 300 nm is applied under the same conditions as in FIG. 13. Image obtained by measuring under a microscope. As can be seen in Figures 13 and 14, it can be seen that the sensitivity of the quantum dot image detection of the cell is much improved when using the optical arrangement structure applying the metal nano grating according to the embodiment.

15 is a schematic exploded perspective view of a biochip to which a metal nano lattice structure is applied to improve sensitivity of fluorescence measurement according to an embodiment of the present invention. The biochip 100 as shown in FIG. 15 is disposed at the position of the metal lattice / film layers 33 and 31 shown in FIG. 1 (ie, placed on the prism 20) and applied to a total reflection fluorescence microscope. In addition, the biochip 100 may be usefully used for bio experiments such as various analysis and testing of biological samples such as cells cultured in the chip. After supplying and circulating a fluid such as a medium solution and a drug to the cell culture chamber 112 through a tube 161 such as a silicon tube connected to the inlet hole 161a, the tube residue connected to the outlet hole 162a ( 162) can be discharged to the outside. As a result, various bio experiments can be carried out in-situ by culturing the cells in the cell culture chamber 112 in the biochip 100 or by introducing a desired drug into the cells. It can be usefully applied as a lab-on-a-chip.

Referring to FIG. 15, the biochip 100 includes a glass substrate 40 made of SF10 or the like and the above-described metal lattice / thin film layers 31 and 33 formed on the glass substrate 40. The metal lattice / thin film layers 31 and 33 include a metal thin film 31 and a metal lattice 33, such as a silver thin film, and the description thereof will be omitted since it is the same as described above with respect to the total reflection microscope 10.

In addition, the biochip 100 further includes a cell culture plate member 102 disposed on the metal lattice / thin film layers 33 and 31. The cell culture plate member 102 is formed with one or more cell culture chambers 112. The cell culture chamber 112 is constituted by a through hole penetrating the plate member 102 and serves as a cell culture medium, and a medium solution containing cells is supplied to the chamber 112. A biological sample, such as a cell, may be cultured and adhered to the metal lattice portion exposed (ie, directly below the chamber) in the chamber 112.

In addition, the biochip 100 may include a microchannel plate member 104 disposed on the cell culture plate member 102. The microchannel plate member 104 is provided with a through opening 124 in communication with the cell culture chamber 112, and the through opening for supplying a fluid such as a medium solution or a drug to the cell culture chamber 112. A micro-fluidic channel 114 connected to 124 is formed.

The metal lattice / thin film layers 33 and 31, the cell culture plate member 102, and the microchannel plate member 104 are stably covered by the lower glass cover plate 101 and the upper glass cover plate 151. Can lose. By fastening the screw 171 coupled to the upper glass cover plate 151 to the hole 103 of the lower glass cover plate 101, the entire structure of the biochip 100 may be assembled. In assembling the entire structure, the plate members must be aligned so that the opening 124 and the chamber 112 are in communication with each other.

In addition, the close contact plate member 106 may be further disposed between the upper glass cover plate 151 and the fine flow path plate member 104. The close plate member 106 may be elastic or cushioned in a thickness direction such as rubber or a soft material such as a silicone polymer material to closely adhere the upper glass cover plate 151 and the fine flow path plate member 104 to each other. It plays a role. A hole 116a is provided in the contact plate member 106 so that the inlet hole 161a for supplying a fluid (medium solution, drug, etc.) formed in the upper glass cover plate 151 can communicate with a portion 114a of the microchannel. ) Is formed. In addition, the contact plate member (not shown) so that the outlet hole 162a for discharging the fluid (circulated medium solution, the residue of the drug, etc.) formed in the upper glass cover plate 151 communicates with a portion 114b of the microchannel. Another hole 116b is formed in 106. Therefore, when the fluid enters the inlet hole 161a through the tube 161, the fluid passes through the hole 116a of the contact plate member 106 to the upper portion 114a of the microchannel and passes the microchannel 114. Split through and enter each through opening 124 and are supplied to the cell culture chamber 112. The remaining fluid used and remaining in the cell culture chamber 112 exits through the through opening 124, the aperture 116b, the outlet aperture 162a and the tube 162.

By using the metal lattice / thin film layers 33 and 31 as described above, cells cultured in the biochip 100 can be detected and observed with higher detection sensitivity through a total reflection fluorescence microscope.

The present invention is not limited by the above-described embodiment and the accompanying drawings. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, .

1 is a diagram schematically showing a total reflection fluorescence microscope according to an embodiment of the present invention.

2 is a cross-sectional view showing an optical arrangement for generating surface plasmon resonance as a main part including a metal nano lattice structure applicable to the total reflection fluorescence microscope of FIG. 1.

3 is a graph showing reflectance according to an incident angle of light (laser light) incident on an optical arrangement structure in embodiments of the present invention.

4 is a view showing the magnetic field strength of the vanishing wave in the total reflection phenomenon for the metal nano lattice having different materials, periods and thickness conditions.

FIG. 5 is a graph showing a profile of magnetic field strength according to a distance from a vanishing wave generating surface, with and without silver nano gratings having the same base period thickness and different grating thickness.

6 is a scanning electron microscopy photograph of the nano-lattice structure according to an embodiment of the present invention.

7 is an atomic force microscopy photograph of the nano-lattice structure according to an embodiment of the present invention.

8 is a graph showing a profile extracted from the atomic force micrograph data of FIG.

9 is a photograph showing a fluorescent microbead measured by a total reflection microscope of the prior art.

10 is a photograph showing a fluorescent microbead measured by a total reflection microscope according to an embodiment of the present invention.

FIG. 11 is an electron scanning micrograph of cells cultured on silver nanogrids in accordance with an embodiment of the present invention. FIG.

12 is an enlarged photograph of the measurement magnification of the photograph of FIG. 11.

FIG. 13 is a photograph obtained by measuring a quantum dot applied to A431 cells with a total reflection fluorescence microscope according to a conventional example. FIG.

14 is a photograph obtained by measuring a quantum dot applied to A431 cells with a total reflection fluorescence microscope according to an embodiment of the present invention.

15 is an exploded perspective view illustrating a biochip to which a metal nanogrid structure according to an exemplary embodiment of the present invention is applied.

20: prism 30: metal lattice / thin layer

31: metal thin film 33: metal lattice

50: optical batch structure 55: sample (sample)

60: light source 62: beam expander

64: polarizer 66: reflecting mirror

70: objective lens 80: fluorescence detection unit

Claims

delete

Glass substrates;

A metal lattice / thin film layer formed on the glass substrate to support a sample including fluorescent molecules and having a nanolattice structure on the top thereof;

A plate member for cell culture disposed on the metal lattice / thin film layer and in which at least one cell culture chamber is formed; And

A micropath plate member disposed on the cell culture plate member and having a through opening communicating with the cell culture chamber and having a microchannel connected to the through opening for supplying a fluid to the cell culture chamber. Include,

The metal lattice / thin film layer,

Metal thin film; And

And a metal lattice formed on the metal thin film to have a nano lattice structure having a constant lattice thickness (d _g ) and a certain lattice period (A) smaller than a wavelength of incident light, thereby generating local surface plasmon resonance for the incident light. Biochip.

The method of claim 8,

The metal grating has a plurality of stripe-like grating structure arranged in parallel to each other on the metal thin film.

The method of claim 8,

And the metal thin film and the metal lattice are formed of the same metal material.

The method of claim 8,

And the metal lattice / thin film is formed of silver or gold.

The method of claim 8,

The grating thickness (d _g ) of the metal grating is 10 to 20 nm, the thickness (d _f ) of the metal thin film is 10 to 20 nm, the grating period (A) of the metal grating is 100 to 300 nm Featuring biochips.

delete

The method of claim 8,

A lower glass cover plate disposed under the glass substrate; And

Further comprising: an upper glass cover plate disposed on the fine flow path plate member,

The upper glass cover plate is formed with an inlet hole for communicating with the microchannel and for supplying a fluid to the cell culture chamber, and an outlet hole for discharging the fluid that has communicated with the microchannel and passed through the cell culture chamber. A biochip, characterized in that formed.

The method of claim 14,

Further comprising a close contact plate member disposed between the upper glass cover plate and the fine flow path plate member, the close contact plate member serves to closely contact the upper glass cover plate and the fine flow path plate member, The close contact plate member has a hole in communication with the inlet hole and the micro channel, and a hole in communication with the outlet hole and the micro channel.